In vivo Imaging and Quantitation of Adoptively Transferred Human Antigen-Specific T Cells Transduced to Express a Human Norepinephrine Transporter Gene

Memorial Sloan-Kettering Cancer Center, New York, New York 10021, USA.
Cancer Research (Impact Factor: 9.33). 01/2008; 67(24):11959-69. DOI: 10.1158/0008-5472.CAN-07-1250
Source: PubMed


Sequential imaging of genetically marked effector cells after adoptive transfer in vivo has greatly enhanced analyses of their biodistribution, growth, and activity both in animal models and in clinical trials of cellular immunotherapy. However, the immunogenicity of cells expressing xenogeneic reporter constructs limits their survival and clinical utility. To address this limitation, we have evaluated a human norepinephrine transporter (hNET) permitting imaging of transduced cells in vivo with a previously approved clinical grade radiolabeled probe, metaiodobenzylguanidine (MIBG). The hNET gene cDNA was cloned from the SK-N-SH cell line and inserted into a bicistronic retroviral vector also encoding green fluorescent protein. Following transfection, human EBV-specific T lymphocytes seemed fully functional in vitro and also selectively accumulated [(123)I]MIBG. In nonobese diabetic/severe combined immunodeficient mice bearing human EBV lymphoma xenografts, as few as 10(4) transduced T cells injected into the tumors could be imaged by single-photon emission computed tomography (SPECT) or positron emission tomography (PET) after i.v. infusion of [(123)I]MIBG or [(124)I]MIBG, respectively. When hNET(+) EBV-specific T cells were infused i.v., their migration and specific accumulation in EBV(+) tumors expressing their restricting HLA allele could be imaged by SPECT or PET over 28 days. Image intensity was closely correlated with the number of T cells accumulated in targeted tumors. The use of two reporter probes (MIBG and 2'-deoxy-2'-fluoro-beta-d-arabinofuranosyl-5-iodouracil) permitted independent contemporaneous tracking of two distinct EBV-specific T-cell subpopulations expressing different reporter genes (hNET-CD4(+) T cells and HSV-TK-CD8(+) T cells) in the same animal using three-dimensional nuclear modalities (SPECT and PET). The hNET-based system described may thus have significant potential as a nonimmunogenic reporter for extended repeated quantitative in vivo imaging of transduced cells in man.

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    • "Continuous in vivo cell tracing over a long period of time can offer valuable information on cellular processes and biomedicinal therapies123456. Among the bioimaging techniques such as positron emission tomography7, single photon emission computing tomography8, and magnetic resonance imaging9, fluorescence technique is advantageous, in terms of more varieties of biocompatible, inexpensive and readily available imaging reagents and more maneuverable instruments that can provide images with higher resolutions at the cellular level1011. "
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    ABSTRACT: Long-term noninvasive cell tracing by fluorescent probes is of great importance to life science and biomedical engineering. For example, understanding genesis, development, invasion and metastasis of cancerous cells and monitoring tissue regeneration after stem cell transplantation require continual tracing of the biological processes by cytocompatible fluorescent probes over a long period of time. In this work, we successfully developed organic far-red/near-infrared dots with aggregation-induced emission (AIE dots) and demonstrated their utilities as long-term cell trackers. The high emission efficiency, large absorptivity, excellent biocompatibility, and strong photobleaching resistance of the AIE dots functionalized by cell penetrating peptides derived from transactivator of transcription proteins ensured outstanding long-term noninvasive in vitro and in vivo cell tracing. The organic AIE dots outperform their counterparts of inorganic quantum dots, opening a new avenue in the development of fluorescent probes for following biological processes such as carcinogenesis.
    Full-text · Article · Jan 2013 · Scientific Reports
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    • "NET mRNA levels appear to correlate in vitro with extent of MIBG uptake [6] [7] [8]. Moreover, a range of cells that do not typically accumulate MIBG can be engineered to do so by transfection of the NET gene [9] [10] [11] [12] [13] [14] [15] [16] [17]. Additional studies in neuroblastoma and other neuroendocrine tumors have suggested that vesicular monoamine transporters (VMATs) and organic cation transporters (OCTs) may also play a role in mediating uptake of MIBG [18] [19] [20]. "
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    ABSTRACT: Purpose. (123)I-metaiodobenzylguanidine (MIBG) is used for the diagnostic evaluation of neuroblastoma. We evaluated the relationship between norepinephrine transporter (NET) expression and clinical MIBG uptake. Methods. Quantitative reverse transcription PCR (N = 82) and immunohistochemistry (IHC; N = 61) were performed for neuroblastoma NET mRNA and protein expression and correlated with MIBG avidity on diagnostic scans. The correlation of NET expression with clinical features was also performed. Results. Median NET mRNA expression level for the 19 MIBG avid patients was 12.9% (range 1.6-73.7%) versus 5.9% (range 0.6-110.0%) for the 8 nonavid patients (P = 0.31). Median percent NET protein expression was 50% (range 0-100%) in MIBG avid patients compared to 10% (range 0-80%) in nonavid patients (P = 0.027). MYCN amplified tumors had lower NET protein expression compared to nonamplified tumors (10% versus 50%; P = 0.0002). Conclusions. NET protein expression in neuroblastoma correlates with MIBG avidity. MYCN amplified tumors have lower NET protein expression.
    Full-text · Article · Sep 2012
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    • "Alternatively, the human dopamine type 2 receptor (D2R) and the somatostatin receptor (SSTr) in conjunction with their respective reporter probes, [18F] FESP and [111In]-DTPA-D-Phe1-octreotide, have been extensively explored [5, 6]. Additional reporter systems include the sodium iodide symporter (NIS) [7] and the human norepinephrine transporter (hNET), which has been recently used to image adoptively transferred human antigen-specific T cells in mice [8]. Each system has its advantages and limitations pertaining to immunogenicity and potential use as a suicide gene (tk), target specificity, uptake levels, background in normal tissues (NIS), and potential for unwanted biological activity (D2R, SSTr, NIS, hNET) discussed in reference [9]. "
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    ABSTRACT: The objective of this article is to develop internalizing positron emission tomography (PET) reporter genes for tracking genetically modified T cells in vivo. The transmembrane and cytoplasmic domains of the human transferrin receptor (TfR) and CD5 were each fused to the carcinoembryonic (CEA) minigene N-A3 and expressed in Jurkat T cells. Internalization was evaluated by confocal microscopy or by intracellular uptake of ¹²⁵I-labeled anti-CEA scFv-Fc. Reporter gene-transfected Jurkat xenografts in mice were analyzed by immunohistochemistry (IHC) and imaged by PET using ¹²⁴I- or ⁶⁴Cu-scFv-Fc as tracers. Surface expression of TR(1-99)-NA3 was lower than that of NA3-CD5. Both reporter genes were internalized following binding of the anti-CEA antibody fragment. IHC of tumors showed strong staining of NA3-CD5, whereas TR(1-99)-NA3 stained weakly. Specific targeting of TR(1-99)-NA3 or NA3-CD5 was shown by PET in xenografted mice. The in vivo imaging studies suggest a potential application of the internalizing form of CEA (N-A3) as a PET reporter gene.
    Full-text · Article · Jun 2011 · Molecular imaging and biology: MIB: the official publication of the Academy of Molecular Imaging
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